1. Field of the Invention
The present invention relates to a method of improving the therapeutic activity of von Willebrand Factor obtained from a material comprising said factor. More particularly, the present invention relates to a method for increasing the therapeutic activity of von Willebrand Factor obtained from a material comprising said factor bound to a monoclonal or polyclonal antibody specific thereto.
In another aspect, the present invention relates to a method of separating, purifying and increasing the therapeutic activity of von Willebrand Factor, and providing an active protein for the treatment of the von Willebrand's disease.
Von Willebrand Factor (hereinafter vWF) circulates in plasma complexed with Factor VIII procoagulant activity protein (hereinafter VIII). The complex is believed to have two biologic functions: vWF corrects defects of platelet function in von Willebrand's disease, and VIII corrects the clotting defect in hemophilia. vWF exists in plasma as a series of multimeric forms of a protein ranging in molecular weight from about 1.times.10.sup.6 to about 20.times.10.sup.6 Daltons. Von Willebrand disease is characterized by the absence or reduced level of the higher molecular weight forms of this protein and is manifest by prolonged bleeding due to the inability of platelets to aggregate and initiate clotting at the wound cite. Traditionally, treatment of bleeding episodes caused by this disease consisted of the administration of cryoprecipitate prepared from human plasma containing normal vWF. This treatment exposed the patient to other coagulation factors and other plasma proteins, particularly fibronectin and fibrinogen. Repeated administration of extraneous proteins has been shown to be deleterious to the health of the patient because of changes brought about in blood viscosity. Treatment with cryoprecipitate also exposed the patient to infectious viruses, such as hepatitis viruses and AIDS viruses which may be present in the donor's plasma.
Commercial concentrates of Factor VIII prepared from plasma by cryoprecipitation of the vWF/Factor VIII complex, followed by purification and concentration, have not been proven to be as effective as expected in the treatment of von Willebrand's disease. The therapeutic inadequacy of these preparations has been in part attributed to the absence of sufficient potency of higher molecular weight forms of vWF that are believed to be essential for the restoration of hemostatis when bleeding occurs in vWF deficient patients. There has been no attempt to treat von Willebrand patients with isolated and purified vWF because the isolation of a biologically active vWF in large quantities could not be carried out with existing technology. With the development of monoclonal and polyclonal antibodies specific to vWF, such technology has become available.
2. Reported Developments
U.S. Pat. No. 4,361,509 and U.S. Pat. RE. 32,011 issued to Zimmerman, et al. disclose a method for the preparation of high purity VIII comprising the steps of:
1. adsorbing the VIII/vWF complex from a plasma or commercial concentrate source onto agarose beads bound to a monoclonal antibody specific to vWF; PA1 2. eluting VIII with a salt solution; PA1 3. adsorbing the eluted VIII on aminohexyl agarose column; and PA1 4. eluting the VIII with a salt solution. PA1 a. adsorbing VIII/vWF complex from concentrate source onto a solid phase backbone containing monoclonal or polyclonal or cocktail of monoclonal anti-vWF antibodies covalently linked to said solid phase backbone; PA1 b. washing unbound proteins from the solid phase; PA1 c. eluting VIII with a chaotropic solution; and PA1 d. eluting vWF from the solid phase with a chaotropic solution.
Immunoadsorbent column described in said references is regenerated by eluting vWF with 3M aqueous sodium thiocyanate (NaSCN) solution. This step results in the preservation of the monoclonal antibody on the column so that the same may be used again in the process. The vWF/sodium thiocyanate solution is discarded as a waste.
The present invention utilizes this waste material as a source of vWF although it is not limited thereto. In the process of developing the invention, it was discovered that biological activity of vWF is normally lost as a result of its elution from the antibody column with sodium thiocyanate solution. Illustrative is the result obtained on material produced according to the teaching of the cited references, description of which follows.
Normal human plasma was collected from normal plasmapheresis donors and diluted with 1/6 volume of 4% sodium citrate. The citrated plasma was immediately frozen and later pooled with additional citrated plasmas and allowed to thaw. Cryoprecipitable proteins were isolated at 0.degree.-2.degree. C. by cold centrifugation of the thawed plasma pool. Approximately 100 g of cryoprecipitate was collected from 100 liters of pooled, citrated plasma. The cryoprecipitate was resuspended at 37.degree. C. in 4 volume of water containing 60 mM glycine and 40 mM sodium chloride at pH 7, then clarified with Al(OH)3 and by centrifugation. Four liters of clarified cryosolution were passed over a 10.times.15 cm column of anti-vWF SEPHAROSE. The nonbound proteins were washed through the column with a solution of lysine (0.1M), histidine (0.02M) and NaCl (0.15M) at pH 7. Factor VIII activity was eluted with 0.25M CaCl.sub.2. The vWF proteins were eluted with 3M NaSCN.
The one liter immunoaffinity column yielded between 500 and 800 ml of thiocyanate solution containing between 170 and 540 mg von Willebrand protein, having antigenic and molecular properties similar to the unpurified von Willebrand protein found in cryoprecipitate. Measurement, using the ristocetin cofactor, platelet agglutination test, however, showed that very little activity was preserved during the elution with 3M NaSCN.
Similar results have been reported by Hornsey, et al. (Thrombosis and Haemostasis--F. K. Schattauer Verlag GmbH, Stuttgart 57 (1) 102-105 (1987).
It is clear that such activity is not quite adequate for therapeutic use and there exists a need for a method for preserving vWF activity in the by-product or waste product obtained by the method of separating and purifying VIII.